The current consensus is that a successful HIV vaccine will need to elicit both neutralizing antibody and cellular immune responses. The antibody response should be broadly neutralizing. The feasibility of eliciting such a response is suggested by the existence of a small panel of broadly neutralizing human monoclonal antibodies (mAbs). These mAbs are valuable tools in vaccine design. They help to define conditions for antibody-mediated protection, they reveal neutralizing epitopes, they provide quality control for vaccine candidates (on which neutralizing epitopes should be expressed) and they lead directly to structures that are vaccine candidates e.g. engineered envelope molecules, peptides and carbohydrates. However, the number of broadly neutralizing mAbs is small and they are all derived from subtype B-infected donors. In order to have a larger panel of mAbs that will more effectively neutralize global HIV-1 isolates and that will stimulate vaccine research, we propose a renewed effort to generate such Abs using phage display, yeast display and memory B cell technologies. This effort is timely given a number of advances including the identification of donors with broad serum neutralizing activity and the refinement of technologies for human mAb generation and selection. Aim 1 is to generate and characterize broadly neutralizing human mAbs from subtype A, B, and C-infected donors. 4E10 is clearly the most broadly neutralizing mAb described to date but its potency is generally only moderate. We have recently solved the structure of Fab 4E10 in complex with its peptide antigen and now propose to apply in vitro evolution techniques to improve the affinity and neutralizing characteristics of 4E10. Aim 2 is to generate affinity-enhanced versions of 4E10 for use as entry inhibitors and as improved templates for immunogen design. Both aims are focused on providing the tools that will eventually allow us to generate or design immunogens that reliably elicit broadly neutralizing Abs.